Pulmonary hypertension is a serious chronic lung disease which regularly leads to death if untreated. The term applies to diseases of various causes, which are characterized by a structural change in the pulmonary vasculature, and in which there is an increase in blood pressure in the pulmonary arterial system to more than 25 mm Hg [1]. This usually results, in affected patients, in stress-dependent shortness of breath, and general loss of capacity. Disease progression leads to a narrowing of vessels resulting from a transformation (remodeling) and thickening of all three layers of the vessel wall, i.e. intima, media and adventitia [2]. This often leads to resting dyspnea, global respiratory insufficiency, and the congestive syndromes associated with right-sided heart failure and in the long-term to heart failure. Pulmonary arterial hypertension is a particularly severe form of pulmonary hypertension in which the median survival from diagnosis is only about three years [3], and diagnosis is often made very late due to the initially mild symptoms.
Several animal models that functionalize different disease characteristics are available for investigating the mechanisms of pulmonary hypertension, and for pre-clinical treatment studies. These include both inducible models (hypoxia, monocrotaline, or antigens, for example) and transgenic models, wherein the selection of a suitable model depends on the research question being examined [4].
Not all possible causes of the various forms of pulmonary arterial hypertension have been explained to date. Nevertheless, there are several well-known and therapeutically relevant factors. For example, in cases of idiopathic pulmonary arterial hypertension, the increased release of vasoconstrictive factors is discussed [5-7], while in many cases of familial pulmonary arterial hypertension, mutations of BMPR2 [8] or the Activin receptor-like kinase 1 (ALK1) gene [9] are considered likely causes.
The development of new therapeutic options for the treatment of pulmonary hypertension or pulmonary arterial hypertension is an urgent need. Such a development could be the transfer of therapeutic genes into lung tissue, and more particularly into the pulmonary endothelium. Vectors that allow a specific and efficient gene transfer into the pulmonary endothelium have not yet been described in the prior art. Gene therapy using viral vectors is a promising treatment option for diseases that do not respond at all, or not adequately, to conventional treatment. This approach is based on the introduction of therapeutic genes into the organism being treated, by means of viruses which have been modified in such a manner that they have the sequence of the corresponding gene in their genome. Viral vectors which have already been used in a gene therapy regimen for gene therapy approaches are based on retroviruses, lentiviruses, adenoviruses and adeno-associated viruses.
Adeno-associated viruses (AAVs) are promising candidates for use in clinical practice because they are classified as relatively safe. AAV vectors are able to introduce a transgene into a tissue and express the gene stably and efficiently in the tissue. At the same time, these vectors have no known pathogenic mechanisms [10]. Of particular importance for clinical use are the AAV vectors of serotype 2 (AAV2), which are considered to be particularly well investigated. After the AAV vectors are introduced, the transgenes can be incorporated in different forms in the transfected cells—for example as episomal, single- or double-stranded DNA. Concatamer forms of the DNA have also been demonstrated in transduced cells.
The genome of AAV2 is formed by a linear, single-stranded DNA molecule of approximately 4700 nucleotides in length and has inverted terminal repeats (ITRs) at both ends. The genome also includes two large open reading frames which are called the replication region (rep) and the capsid region (cap). The replication region encodes proteins that are required as part of the virus replication. The capsid region, however, encodes for the structural proteins VP1, VP2 and VP3, which make up the icosahedral capsid of the virus.
Like most vectors which have gene therapy applications and are known in the prior art, however, wild-type AAV vectors, such as the AAV2 vectors described above, do not possess sufficient specificity for a particular tissue, and infect a wide variety of cell types. As such, systemic administration of wild-type vectors leads to insufficient transduction of lung tissue, and severe immune reactions are expected in the treatment subject due to the unwanted transduction of other tissues. Progress in the development of viral vectors which have an increased specificity for particular organs has been made in the past by the use of peptide ligands, which are able to direct the vectors to a particular organ [11-12]. It has been shown that certain peptide ligands bring about a “homing” to various organs such as the brain.
Reading [13] describes a method which enables the screening for tropism-modified capsids of AAV2 in randomized peptide libraries. From these libraries, vectors can be isolated which specifically transduce a desired cell type in vitro. However, it has been surprisingly found that capsids selected in this manner are often unsuitable for use in vivo because they lack the necessary specificity in animal models [14].
There remains a great need for agents that are able to modulate the tropism of viral vectors and thus ensure adequate cell or tissue specificity to enable targeted delivery of a viral vector into the lung. Such vectors enable specific expression of therapeutic genes in lung tissue, for the corresponding, effective treatment of diseases and/or disorders of the lungs.
The present invention makes available viral vectors for targeted gene transfer to the lungs. The viral vectors according to the invention express on their capsid surface a previously unknown amino acid sequence that is specifically recognized in vivo by receptors on the endothelial tissue of the lung. As such, the viral vectors of the present invention specifically transduce the lung tissue of a patient following systemic administration to the same.
The viral vectors according to the invention also enable a strong and persistent expression of a transgene in the endothelial cells of the lung with only minor immune response, and are therefore particularly suitable for gene therapy treatments of certain pulmonary disorders and/or lung diseases. After transfection, the AAV vectors only instigate a minor immune response in the host and are therefore particularly suitable for gene therapy.